Hcd56

Manufactured by BioLegend

Sourced in United States

The HCD56 is a lab equipment product. It is designed for use in life science research applications. The core function of the HCD56 is to facilitate the detection and analysis of specific biological molecules or cellular components.

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Lab products found in correlation

Close spelling variants of this product

CD56 (HCD56; (9 citations) Anti-CD56 (HCD56, (7 citations) CD56-PE-Cy7 (HCD56, (7 citations) CD56 (clone HCD56; (5 citations) CD56-PE (clone HCD56, (4 citations) Clone HCD56; (4 citations) CD56-BV605 (HCD56; (3 citations) Anti-CD56 (clone HCD56; (3 citations) Anti-CD56-AF488 mAb (clone HCD56, (3 citations) PE/Cy7-conjugated CD56 (HCD56; (3 citations)

18 protocols using hcd56

Comprehensive Flow Cytometry for Tumor Immune Profiling

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Single cell suspensions of tumor and tumor-adjacent tissue were stained
with surface antibodies for CD19, CD20, CD3, CD8, CD4, CD56 and CD11c (clones
HIB19, 2H7, HIT3a, RPA-T8, RPA-T4, HCD56, and 3.9, respectively, Biolegend) and
an intracellular stain for the transcription factor FoxP3 (clone PCH101,
Ebioscence) for Figure 1 and for CD19, CD20, CD21, CD69, CD27 and HLA-DR (clones HIB19, 2H7, HB5, FN50, O323, and L243,
respectively, Biolegend and Ebioscience) for Figs. 2 and 3. Positive and negative
controls for staining were PBLs from patients or control donors, lymphocytes
from control donor spleens, lymphocytes from control (disease-free) lungs,
lymphocytes from NSCLC pleural metastases, and PBL from untreated,
HIV⁺ patients. Flow cytometry was conducted using an LSR
II (BD Biosciences) and data were analyzed using FlowJo software (Tree Star,
Inc.). For Fig. 2B, total lymphocyte infiltration was calculated using the following equation: [Total cell
number from tissue X % lymphocyte gate X % lymphocyte
population, i.e. CD19⁺]/ [mass of tissue in
grams].

Bruno T.C., Ebner P.J., Moore B.L., Squalls O.G., Waugh K.A., Eruslanov E.B., Singhal S., Mitchell J.D., Franklin W.A., Merrick D.T., McCarter M.D., Palmer B.E., Kern J.A, & Slansky J.E. (2017). Antigen-presenting intratumoral B cells affect CD4+ TIL phenotypes in non-small cell lung cancer patients. Cancer immunology research, 5(10), 898-907.

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Isolation and Characterization of Tumor-Associated Macrophages from Clear Cell RCC

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Clear cell RCC patient tissue specimens were prepared by mechanical disruption using a razor blade followed by treatment with 280 U/mL Collagenase Type 3 (Worthington Biochemical) and 4 ug/mL DNase I (Sigma) at 37°C for 1 hour with periodic vortexing. Digested tissues were passed through 70 um filters. Resulting cells were resuspended in 44% Percoll – 66% Percoll gradient (Sigma) and centrifuged for 30 minutes at 1,900 g with no brake. Mononuclear cells were collected and immediately stained for flow cytometry analysis following Fc blocking (company) and live/dead staining (company). The antibodies used for flow cytometry were: HLA-DR (L243, Biolegend #307618), CD14 (HCD14; Biolegend #325608), CD45 (2D1; eBioscience 11–9459-42), CD16 (3G8; Biolegend 302008), CD56 (HCD56; Biolegend 318318), and CD3 (7D6; Invitrogen MHCD0317). Tumor-associated macrophages (TAM) were identified as CD45⁺CD3^–Lin^-HLA-DR⁺CD14⁺CD16⁺. Data are reported as the percent of all cells.

Hakimi A.A., Voss M.H., Kuo F., Sanchez A., Liu M., Nixon B., Vuong L., Ostrovnaya I., Chen Y.B., Reuter V.E., Riaz N., Cheng Y., Patel P., Marker M., Reising A., Li M., Chan T.A, & Motzer R.J. (2019). Transcriptomic Profiling of the Tumor Microenvironment Reveals Distinct Subgroups of Clear Cell Renal Cell Cancer - Data from a Randomized Phase III Trial. Cancer discovery, 9(4), 510-525.

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Immune Checkpoint Inhibitor Binding Assay

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Fresh PBMCs (1x10⁵/condition) were incubated with nivolumab (InvivoGen, 375938995), pembrolizumab (InvivoGen, 375939001) or IgG₄ LEAF isotype (BioLegend, 403702) in 96‐well round‐bottom plates (GreinerBioOne) in a total of 100 μl of PBS containing 10 pg/ml to 100 μg/ml. After 30 min at 4°C, cells were washed three times with PBS (150 μl/well), then incubated with RG7769 (10 μg/well in PBS). After 30 min at 4°C, cells were again washed three times with PBS (150 μl/well), then stained with 50 μl of master mix containing CD45‐PerCPCy5.5 (1:100, HI30, BioLegend, 304028), CD4‐V500 (1:100, RPA‐T4, BD Horizon, 560768), CD56‐APC (1:100, HCD56, BioLegend, 318310), CD16‐APC (1:100, 3G8, BD PharMingen, 561248), CD8‐BV421 (1:100, RPA‐T8, BioLegend, 301036) and CD3‐AF488 (1:100, SP34‐2, BD PharMingen, 557705) and anti‐PGLALA‐PE (M.1.7.24, 1:160). Cells were analyzed on a BD FACS‐Symphony cytometer (five lasers: 355, 405, 488, 561, 637 nm). Compensation was performed using VersaComp antibody capture beads (Beckman, B22804).

Junker F., Gulati P., Wessels U., Seeber S., Stubenrauch K., Codarri‐Deak L., Markert C., Klein C., Camillo Teixeira P, & Kao H. (2021). A human receptor occupancy assay to measure anti‐PD‐1 binding in patients with prior anti‐PD‐1. Cytometry, 99(8), 832-843.

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Assessing HA-Directed B Cell Binding

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In order to assess HA- directed B cell binding and cross-reactivity, peripheral blood mononuclear cells (PBMC’s) from samples with different stem binding based on ELISA assay (n = 18) were further analyzed by flow cytometry. Three groups were selected for the analysis: negative for stem antibodies (n = 5), reactive to one stem only (n = 4), and samples with cross-reactive stem binding (n = 9). Cryopreserved PBMC’s were thawed in R-10 media containing 50 U/mL of Universal Nuclease (ThermoFisher). Cells were washed and resuspended in PBS for staining with UV-Blue viability dye (ThermoFisher) for 20 minutes at room temperature. After washing, surface staining was performed using antibodies against IgM (G20-127, BD Biosciences), IgG (G18-145, BD Biosciences), CD8 (RPA-T8, BioLegend), CD3 (OKT3, BioLegend), CD56 (HCD56, BioLegend), CD14 (M5E2, BioLegend), CD19 (J3-119, Beckman Coulter), CD27 (O323, BioLegend), CD38 (HIT2, BioLegend), and HA probes^{27 –29 (link)}. H1 NC 99 and H5 INDO 05 HAs were expressed and biotinylated followed by fluorochrome labeling as previously described^{29 (link)}. Stained cells were run on a BD LSRFortessa X-50 and data analysis was performed using FlowJo (TreeStar). The gating strategy is demonstrated in supplemental Fig. 2. Statistical significance at 95% confidence interval was done using two-tailed t-test.

Yassine H.M., McTamney P.M., Boyington J.C., Ruckwardt T.J., Crank M.C., Smatti M.K., Ledgerwood J.E, & Graham B.S. (2018). Use of Hemagglutinin Stem Probes Demonstrate Prevalence of Broadly Reactive Group 1 Influenza Antibodies in Human Sera. Scientific Reports, 8, 8628.

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Isolation of Monocyte Subsets

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For the isolation of classical and non‐classical monocytes from the peripheral human blood, PBMCs were incubated with specific antibodies against CD14 (PE, HCD14), CD16 (APC/Cy7, 3G8), CD56 (FITC, HCD56, all BioLegend), CD3 (VioGreen, REA613) and CD19 (VioBlue, LT19, both Miltenyi) and sorted on Astrios EQ Sorter (Beckman Coulter).

Sommer K., Heidbreder K., Kreiss L., Dedden M., Paap E., Wiendl M., Becker E., Atreya R., Müller T.M., Atreya I., Waldner M., Schürmann S., Friedrich O., Neurath M.F, & Zundler S. (2023). Anti‐β7 integrin treatment impedes the recruitment on non‐classical monocytes to the gut and delays macrophage‐mediated intestinal wound healing. Clinical and Translational Medicine, 13(4), e1233.

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Flow Cytometric Profiling of PBMCs

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Frozen PBMCs were thawed, washed in FACS buffer (2% FBS, 1mM EDTA in PBS), and counted on TC20 (Biorad) before surface staining using two independent flow panels. For the innate panel, the following antibodies were used: CD3 (SP34, BD Pharmingen) and CD20 (2H7, BioLegend) for the exclusion of T & B lymphocytes, respectively. We further stained for CD56 (HCD56, Biolegend), CD57 (HNK-1, BioLegend), KLRG1 (SA231A2, BioLegend) CD16 (3G8, BioLegend), CD14 (M5E2, BioLegend), HLA-DR (L243, BioLegend), CD11c (3.9, ThermoFisher Scientific), CD123 (6H6, BioLegend) and CD86 (IT2.2, BioLegend). For the adaptive panel, the following antibodies were used: CD4 (OKT4, BioLegend), CD8b (2ST8.5H7, Beckman Coulter), CD45RA (HI100, TONBO Biosciences), CCR7 (G043H7, BD Biosciences), CD19 (HIB19, BioLegend), IgD (IA6–2, BioLegend), CD27 (M-T271, BioLegend), KLRG1 (SA231A2, BioLegend) and PD-1 (Eh12.2h7, BioLegend). Cells were stained with Ghost Dye viability dye (TONBO biosciences) for 30 minutes at 4C per manufacturer’s instructions, washed, surface stained with either innate or adaptive panels for 30 minutes at 4C. Samples were then washed and analyzed on Attune NxT Flow Cytometer (ThermoFisher Scientific, Waltham MA).

Sureshchandra S., Zulu M.Z., Doratt B., Jankeel A., Tifrea D., Edwards R., Rincon M., Marshall N.E, & Messaoudi I. (2021). Deep immune profiling of the maternal-fetal interface with mild SARS-CoV-2 infection. bioRxiv.

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Flow Cytometric Immunophenotyping of Hematopoietic Progenitor Cells

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Cells were thawed at 37 °C, washed with sterile PBS and incubated with biotinylated anti-human lineage antibodies directed against CD2 (clone RPA-2.10, BioLegend), CD3 (HIT3a, 300304, BioLegend), CD4 (RPA-T4, BioLegend), CD7 (124-1D1, eBioscience), CD8a (RPA-T8, BioLegend), CD10 (SN5c, eBioscience), CD11b (ICRF44, BioLegend), CD14 (HCD14, BioLegend), CD19 (HIB19, BioLegend), CD20 (2H7, eBioscience), CD56 (HCD56, BioLegend) and GPA (HIR2, BioLegend) and LIVE/DEAD Fixable Aqua Dead Cell Stain (L-34957, Life Technologies). For gating on hematopoietic progenitor cells, this was followed by secondary staining with anti-human CD34–APC (8G12, BD Biosciences), anti-CD38–PE/Cy7 (HIT2, BioLegend), anti-CD90–FITC (5E10, BD Biosciences), anti-CD45RA-PB (MEM-56, Thermo Fisher Scientific), anti-CD123–PE (7G3, BD Biosciences) and streptavidin-APC/Cy7 (BioLegend). Intracellular staining with anti-human Ki-67–BV605 antibody (BioLegend) was performed using the BD Cytofix/Cytoperm Fixation/Permeabilization kit. Samples were run on an LSR II flow cytometer (BD Biosciences), and recorded events were analyzed with FlowJo 10 software (BD). Individual fluorescence-minus-one controls were used to determine gating. HSCs were identified as lineage⁻CD34⁺CD38⁻CD45RA^loCD90⁺ cells, CMPs as lineage⁻CD34⁺CD38^intCD45 RA⁻CD123^int cells and GMPs as lineage⁻CD34⁺CD38^intCD45RA⁺CD123^int cells.

Rohde D., Vandoorne K., Lee I.H., Grune J., Zhang S., McAlpine C.S., Schloss M.J., Nayar R., Courties G., Frodermann V., Wojtkiewicz G., Honold L., Chen Q., Schmidt S., Iwamoto Y., Sun Y., Cremer S., Hoyer F.F., Iborra-Egea O., Muñoz-Guijosa C., Ji F., Zhou B., Adams R.H., Wythe J.D., Hidalgo J., Watanabe H., Jung Y., van der Laan A.M., Piek J.J., Kfoury Y., Désogère P.A., Vinegoni C., Dutta P., Sadreyev R.I., Caravan P., Bayes-Genis A., Libby P., Scadden D.T., Lin C.P., Naxerova K., Swirski F.K, & Nahrendorf M. (2021). Bone marrow endothelial dysfunction promotes myeloid cell expansion in cardiovascular disease. Nature cardiovascular research, 1(1), 28-44.

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Multiparameter FACS Staining for Immune Profiling

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For FACS staining, we used the following antibodies to target: CD16 (FITC; eBioCB16), CD14 (PE-Cy7; 61D3), CD15 (APC; MMA), IL-4 (APC; 8D4-8), and IFN-γ (4S.B3)—all purchased from eBioscience (San Diego, CA, USA)—and CD11b (FITC; ICRF44), CD56 (PE; HCD56), CD33 (PE; WM53), IL-10 (PE; JES3-9D7) CD45 (APC; HI30), CD163 (APC; GH1/61), CD3 (PE-Cy7; HIT3a), CD4 (FITC; OKT4), and IL-17A (PE; BL168)—all purchased from BioLegend (San Diego, CA, USA).

Kasper M., Walscheid K., Laffer B., Bauer D., Busch M., Wildschütz L., Wang B., Loser K., Vogl T., Grajewski R.S., Langmann T, & Heiligenhaus A. (2018). The Phenotype of Monocytes in Anterior Uveitis Depends on the HLA-B27 Status. Frontiers in Immunology, 9, 1773.

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Immunophenotyping of PBMC Subsets

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Peripheral blood mononuclear cells (PBMCs) were Fc receptor blocked, labelled with fluorescent antibodies specific for: CD3 (OKT3), CD4 (OKT4), CD8 (HIT8a), CD14 (HCD14), CD19 (HIB19) and CD56 (HCD56; all antibodies were from Biolegend) and dead cells were identified by DAPI exclusion. CD4⁺, CD8⁺, CD14⁺, CD19⁺ and CD56⁺ fractions were collected (Influx cell sorter, BD Biosciences) directly into ice-cold FACS buffer, immediately frozen on dry ice and stored at –80°C.

Macartney-Coxson D., Cameron A.M., Clapham J, & Benton M.C. (2020). DNA methylation in blood—Potential to provide new insights into cell biology. PLoS ONE, 15(11), e0241367.

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Cytokine Production in iNK Cells

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iNK cells were incubated with or without PMA/ionomycin (MULTISCIENCES), K562 tumor cells (E:T = 1:2), or recombinant human IL-2 (1000 IU/mL, Miltenyi) for 2 h, followed by adding BFA/Monensin (MULTISCIENCES) for additional 2-h incubation. Cells were stained with anti-human CD45 (Biolegend, HI30), CD3 (Biolegend, HIT3a), and CD56 (Biolegend, HCD56) antibodies. FIX & PERM Kit (MULTISCIENCES) was used for fixation and permeabilization, followed by intracellular staining for IFN-γ (Biolegend, 4 S.B3) or TNF-α (Biolegend, MAb11). The cells were analyzed with BD LSRFortessa X-20 cytometer (BD Biosciences).

Huang D., Li J., Hu F., Xia C., Weng Q., Wang T., Peng H., Wu B., Wu H., Xiong J., Lin Y., Wang Y., Zhang Q., Liu X., Liu L., Zheng X., Geng Y., Du X., Zhu X., Wang L., Hao J, & Wang J. (2022). Lateral plate mesoderm cell-based organoid system for NK cell regeneration from human pluripotent stem cells. Cell Discovery, 8, 121.

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